1. Field of the Invention
This invention relates to and has among its objects novel methods and compositions for performing an assay for the determination of an analyte in a whole blood sample. The invention finds particular use in immunochromatographic techniques.
The determination of analytes in blood samples has become increasingly more important to medicine, both in diagnosis and therapy. Red blood cells in the sample must be separated prior to performing the assay, which assay is generally performed on blood serum. This is particularly true in assays involving labels, such as enzyme label immunoassays, particularly where a quantitative measurement is to be made. In such determinations one is normally dealing with variable concentrations of the analyte and is normally detecting the difference between two different low level concentrations of analyte. It is a necessary practice to separate red blood cells from the whole blood sample because the cells may inhibit binding which occurs between the sbp members. Furthermore, the cells have enzyme activity which can interfere with the signal produced. Since the quantitative determinations require high precision, the background interference in the assay produced by the presence of red blood cells cannot be tolerated.
An enzyme immunoassay carried out on a whole blood sample thus involves two steps. In the first step, red blood cells are separated from the serum in the whole blood sample. This separation generally involves a centrifugation where the cells settle at the bottom and the serum may be separated by decantation or other method. The separation of the red blood cells from the serum is carried out generally at the site at which the whole blood sample is taken. Following the separation, the serum is transferred to an assay area. Because of the separation step involved, it is necessary to take a relatively large sample of whole blood from a patient, generally on the order of approximately 0.1 to 5.0 ml. Since the taking of the whole blood sample is an invasive procedure, many doctors are reluctant to order assays on a routine basis. A further complicating problem is that most medical offices do not have a blood separator on site. Consequently, the sample must be transferred to an area where the separation of red blood cells from plasma can take place.
There is, therefore, a need for an assay method which can be applied to a whole blood sample of small volume. Such an assay should not have a separate step for removal of red blood cells and should be applicable to a small volume, such as a pin prick drop, of whole blood sample taken from a patient. Such an assay method would allow doctors and others to carry out assays for analytes on a more routine basis
2. Description of the Prior Art
Anderson, Anal. Biochem. (1970) 38:175-189 describes the use of cellulose wicks to monitor agglutination reactions. An enzyme chromatographic immunoassay is disclosed in U.S. patent application No. 398,505, filed July 15, 1982, U.S. Pat. No. 4,435,504. Determination of analytes in a particle-containing medium is described in U.S. patent application No. 519,300, filed July 29, 1983, U.S. Pat. No. 4,552,839.
U.S. Pat. No. 4,168,146 describes an immunoassay employing immunochromatography with antigens followed by contacting the immunochromatograph with an aqueous solution containing labeled antibodies. U.S. Pat. No. 4,233,402 describes a homogeneous assay method employing a combination of enzymes, where the substrate of one enzyme is the product of another. Enhanced production of the product is related to the amount of analyte in the assay medium. U.S. Pat. No. 4,275,149 describes the use of particles where combinations of enzymes may be employed, where the presence of the particles enhances the interaction between two enzymes, where the product of one enzyme is the substrate of the other. Enhanced production of the final product due to the presence of the two enzymes bound to the particle as a result of binding of specific binding pair members is related to tne amount of analyte in the assay medium.
Patents concerned with various immobilized reagents on different types of test strips include U.S. Pat. Nos. 3,993,451; 4,038,485; 4,046,514; 4,129,417; 4,133,639l and 4,160,008. Patents disclosing a variety of methods involving separations of bound and unbound antigen include U.S. Pat. Nos. Re. 29,169; 3,949,064; 3,984,533; 3,985,867; 4,020,151; 4,039,652; 4,067,959; 4,108,972; 4,145,408; and 4,168,148.